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7.9 Appendix 1A-Brain Sampling Procedures

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Entire heads may be submitted fresh or frozen to an approved laboratory.

Obex Harvesting Technique

Recommended tools:

1) obex removal spoon and
2) rat tooth forceps

Additional tools that you may choose to use:

3) scalpel
4) scissors

Obex removal spoon and knife may be fashioned from the "Zwilling J.A. Henkle - Jessica Grapefruit spoon and knife." Alternatively, any butter knife or suitably long and concave spoon (6 mm concavity) can be modified. The edges of the knife must be sharpened. The edges of the spoon should be cut down to measure 22–26 mm at the widest point and sharpened to a blade.

With the aid of an autopsy knife, disarticulate the head at the level of the atlanto-occipital articulation. Place the head dorsal side down on a table with the foramen magnum (the opening of the spinal canal) facing you.

Using the forceps with your left hand, grasp the dura mater (the thick lining around the spinal cord) and with the scissors in your right hand make a single cut down the centre line to form two flaps (this step is not mandatory). Remove the congealed blood from around the cord.

With the forceps in your left hand holding the dura, sever the cranial nerves from the cord. This can be done with scissors or with the obex knife by passing the knife around the cord, keeping the flat side of the knife against the cord (see next picture for position of obex knife with relationship to the cord). This is the most important step in freeing up the cord. The cord must be completely free from attachments (by cranial nerves) in all directions. If you do not sufficiently free the cord up, you will not be able to sample anteriorly enough to get the obex, or one side will be missing.

	[D]

Insert the obex spoon face down into the vertebral canal and move it forward lodging the tip as far cranially as it will go. This should position the tip of the knife over the part of the cord just anterior to the obex and just behind the cerebellar attachments.

	[D]

Pick up the head and (if right handed) hold in left hand, dorsal side down. While pushing down with index finger, manoeuver the tip of the obex knife back and forth over the cord to sever it from the rest of the brain just rostral to the obex and caudal to the cerebellar attachments. Rotate the skull with your other hand in the opposite direction to the knife, to facilitate the cutting of the cord.

Use the obex knife to gently pull the severed portion of the brain from the canal. The obex is recognizable as a V-shaped depression. This portion is the single most important site for diagnostic testing.

Contact the laboratory to which you will be submitting the samples and request information on preparing the sample for submission (fresh frozen or formalinized).

Freeze samples and batch ship in frozen state to an approved laboratory for enzyme-linked immunosorbent assay (ELISA) testing.

Alternatively, place the brain stem and cerebellum in a 500 ml glass jar containing 10% buffered formalin fixative. The ratio of the volume of fixative to tissue should be 10:1. Fix the specimen for 24 hours - 5 days depending on the size of the sample (penetration of formalin is at least 5 mm of tissue thickness/day) and then ship to an approved laboratory. These tissues will be subject to histopathology and immunohistochemistry.

Tissues for scrapie surveillance testing may be packaged and shipped as non-regulated substances in accordance with the Transportation of Dangerous Goods Regulations. The basic four parts packaging system may be used. Following fixation the specimen must be packaged in primary watertight receptacle (whirl pak bag) with labelling. Enclosed in second durable watertight packaging (whirl pak bag) with adequate absorbent material to absorb all fluid in case of breakage. Outer packaging, such as a fibreboard box, protects the contents from external physical damage. The description of the contents on the shippers way bill should include the notation "non hazardous, unregulated, sample."

7.9 Appendix 1B-Sampling Procedures for Genotyping

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Venipuncture

To facilitate the rapid collection of blood samples from multiple sheep in a minimum of time, it is suggested to not waste time "up ending" the sheep. Restrain sheep for venipuncture in a standing position. Do not clip wool.

Occlude thoracic inlet, palpate or approximate the location of the jugular vein and insert vacutainer needle.

Single use needle is required for each animal to prevent cross contamination of deoxyribonucleic acid (DNA).

Collect a full 7-10 mL of blood in an ethylenediaminetetraacetate (EDTA) tube (lilac/purple top).

Make sure that the sample and individual animal identification are irrefutably connected.

Samples should be stored at 4°C until shipped. Ship to laboratory as soon as possible.

Check with individual laboratory for preferences on sample submission and preferred day of submission.

Tissue Samples for Genotyping of Deadstock

Fresh or frozen tissues such as brain, liver, spleen, or kidney can be used. Check with individual laboratory preference for tissue selection. As the head or brain sample is being forwarded for scrapie testing, it would be easiest for the laboratory to forward a sample of brain for genotyping.

7.9 Appendix 1C-3^rd Eyelid Lymphoid Follicle Sampling Procedures

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As potentially infective lymphoid tissue is being harvested and the site of harvesting is a potential route of introduction (eye) of the scrapie agent, precautions are required to prevent the potential transmission of scrapie when sampling potential source flocks. Instruments may be used and discarded after use on a single animal. Alternatively, the forceps and scissors used on an individual sheep are placed in a bag accompanied by the individual sheep identification. These instruments are then stored until the results of the tests are known. If the test results for the individual sheep on which the particular set of instruments were used are positive, it is recommended that those instruments be discarded. If the test results for the individual sheep on which a particular set of instruments were used are negative, those instruments may be cleaned and returned to use.

Technique

One person is required to restrain the sheep.

Verify by checking individual identification that this animal has been genotyped as a QQ171.

Instill several drops of a local ophthalmic anaesthetic agent (proparacaine, lidocaine or xylocaine) into the selected eye.

Apply a drop of 1% histamine solution (can be compounded by veterinary pharmacy, e.g. Veterinary Pharmacy, Imperial Road, Guelph, ON (519) 824-7887 or Strathcona Prescription Centre 8225-105 Street, Edmonton, AB T6E 4H2 (780) 432-0394, (888) 433-2334) to the bulbar surface of the third eyelid. This will dramatically facilitate the visualization of the now hyperemic, slightly raised/irregular area that represents the lymphoid follicles.

Allow 5-6 minutes for applied drops to take effect.

Grasp the edge of the 3^rd eyelid with tissue forceps, evert, and visualize the ventral (bulbar) surface of the 3^rd eyelid.

Using scissors (metzenbaum preferred), blades flush with the 3^rd eyelid, cut identified lymphoid follicles from the 3^rd eyelid. Place sample in test tube or small vial of formalin. Forward sample to laboratory capable of testing lymphoid tissues for scrapie.

7.9 Appendix 1D-Listing of Laboratories

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Canadian Laboratories Providing Scrapie Genotyping (not currently CFIA approved)

Agricultural Genomics Laboratory
Haley Institute of Animal Science and Aquaculture
58 River Road
Nova Scotia Agricultural College
Truro, NS
Canada, B2N 5E3
Phone: 902-893-8126
Fax: 902-895-6734

Bova-Can Laboratories
Saskatchewan Research Council
125-15 Innovation Boulevard
Saskatoon, Sask.
Canada, S7N 2X8
Phone: 306-933-7700
Fax: 306-933-5505

TransBIOTech
201, route Monseigneur-Bourget
Lévis, QC G6V 9V6
Phone: 418-833-8876
Fax: 418-833-8867

Vita-Tech
1345 Denison Street,
Markham, ON L3R 5V2
Phone: 416-798-4988; Toll-free: 800-667-3411
Fax: 905-475-7309

CFIA Laboratories Approved for Scrapie Genotyping

Animal Health Laboratory
Laboratory Services (University of Guelph)
College of Veterinarians of Ontario
Building 49, Box 3612
Guelph, ON N1H 6R8
Phone: 519-824-4120, Ext. 54544
Fax: 519-821-8072

Biogenetic Services Inc.
801, 32^nd Avenue
Brookings, SD 57006
U.S.A.
Contact: Alex Kahler
Phone: 605-697-8500
Fax: 605-697-8507

BioServe Biotechnologies Ltd.
1050 West Street
Laurel, MD 20707
U.S.A.
Contact: Dr. Terri Lehman
Phone: 301-470-3362
Fax: 301-470-2333

Diagnostic Center for Population and Animal Health
Michigan State University
Room A45, Veterinary Medical Center
E. Lansing, MI 48824
U.S.A.
Contact: Roger Maes, DVM, PhD
Annabel Wise, DVM, PhD
Mr. Dan Taylor, MS
Phone: 517-353-2296
Fax: 517-353-4426

GeneCheck Inc.
1629 Blue Spruce Drive, Suite 106
Ft. Collins, CO 80524
U.S.A.
Contact: Ms. Tresea Mulqueen
Phone: 800-822-6740
Fax: 970-472-9956

GenMARK
(Vita-Tech Laboratories, LLC)
1825 Infinity Drive
DeForest, WI 53532
U.S.A.
Contact: Ms. Lynette Childs
Phone: 608-846-0580; Toll-free: 877-766-3446
Fax: 608-846-0584

GeneSeek Inc.
4711 Innovation Drive
Lincoln, NE 68521
U.S.A.
Contact: Mr. Abe Oommen
Phone: 402-435-0665
Fax: 402-435-0664

HAP Typing Facility
Genaissance Pharmaceuticals
Five Science Park
New Haven, CT 06511
U.S.A.
Contact: Jeffrey M. Otto, PhD
Phone: 203-786-3546
Fax: 203-786-3699

Veterinary Genetics Laboratory
School of Veterinary Medicine
University of California
One Shields Avenue
Davis, CA 95616-8744
U.S.A.
Contact: Customer Service
Phone: 530-752-7383
Fax: 530-752-3556

Orchid Helix (tests done in UK)
635 Columbia Street
New Westminster BC V3M 1A7
Phone: 604-523-2945; Toll-free: 800-563-4363
Fax: 604-523-2974

Labogéna
Domaine de Vilvert
78352 Jouy-en-Josas Cedex
France
Phone: 01 34 65 21 21
Fax: 01 34 65 20 51

Laboratories Approved by CFIA for Scrapie Testing

Alberta Agriculture
Agri-Food Systems Branch
104 O. S. Longman Building
6909 - 116 Street
Edmonton AB T6H 4P2
Phone: 780-422-1923
Fax: 780-422-3438

Animal Health Laboratory
Laboratory Services (University of Guelph)
College of Veterinarians of Ontario
Building 49, Box 3612
Guelph ON N1H 6R8
Phone: 519-824-4120, Ext. 54544
Fax: 519-821-8072
Laboratory submission form

Prairie Diagnostic Services
4840 Wascana Parkway
Regina SK S4S 7J6
Phone: 306-787-6435
Laboratory submission form

Laboratoire d'expertises en pathologie animale du Québec
2700 Einstein Street, local C.RC. 242
Ste-Foy QC G1P 3W8
Phone: 418-643-6140
Fax: 418-644-4532

Manitoba Agriculture, Food and Rural Initiatives
Livestock Knowlegde Center
Farm Production Extension
545 University Crescent
Winnipeg, MB R3T 5S6
Phone: 204-945-4174
Fax: 204-945-8062

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